Summary/Abstract Lymphangiosarcoma (LAS) is angiosarcoma with lymphatic differentiation that originates from the malignant transformation of endothelial cells (ECs). The etiology of LAS is largely unknown, although lymphatic malformation (e.g. chronic lymphedema in breast cancer patients) has been recognized as a risk factor for the disease. The long-term goal of the proposed studies is to understand the molecular and cellular mechanisms of lymphatic malformation and progression to LAS in order to develop new strategies for effective therapies of this deadly disease. In the previous funding period, we created a mouse model with inducible EC-specific deletion of Tsc1 which recapitulates salient features of human LAS, and showed that hyper-activation of mTORC1 and increased VEGF autocrine signaling in ECs were required for initiation and maintenance of LAS. In further preliminary studies, we discovered that maintenance of hyper-activation of mTORC1 in TSC-deficient cells required autophagy under glucose-starvation, but not amino acid-starvation or normal conditions. Moreover, autophagy maintains elevated levels of ATP specifically through lipid catabolism and generation of fatty acids as the main source of ATP under these energy stress conditions. In addition, consistent with clinical findings that malignant transformation of vascular malformation to angiosarcoma was accompanied with secondary mutations, preliminary studies employing whole exome sequencing (WES) of LAS samples from our Tsc1i?EC mice model identified secondary mutations in several genes including Cdk6, which is involved in cell cycle regulation and implicated in other cancers. Based on these strong preliminary studies and using our unique novel mouse models, we propose to 1) determine the mechanisms of autophagy in the regulation of lipolysis to produce fatty acids as an alternative fuel for ATP production to maintain mTORC1 hyper-activation in Tsc1-null vascular tumor cells; 2) examine the role of autophagy-mediated lipid catabolism in maintaining mTORC1 hyper-activation in lymphatic malformation and LAS in vivo and evaluate potential therapeutic efficacy of targeting autophagy-mediated lipolysis in mouse and PDX models of lymphatic malformation and LAS; and 3) explore the roles and mechanisms of secondary mutations in Cdk6 and other genes in the development of lymphatic malformation and progression to LAS. Together, the proposed studies to examine reverse regulation of mTORC1 by autophagy through novel mechanisms of lipid metabolism as well as secondary mutations that synergizing with mTORC1 signaling will provide significant mechanistic insights into the disease, which may contribute to novel therapies for this devastating disease.